As part of an ongoing program to develop two-photon (2-) photodynamic therapy (PDT) for treatment of wet-form age-related macular degeneration (AMD) and other vascular pathologies, we have evaluated the reciprocity of drug-light doses in focal-PDT. We targeted individual arteries in a murine window chamber model, using primarily the clinical photosensitizer Visudyne/liposomal-verteporfin. Shortly after administration of the photosensitizer, a small region including an arteriole was selected and irradiated with varying light doses. Targeted and nearby vessels were observed for a maximum of 17 to 25 h to assess vascular shutdown, tapering, and dye leakage/occlusion. For a given end-point metric, there was reciprocity between the drug and light doses, i.e., the response correlated with the drug-light product (DLP). These results provide the first quantification of photosensitizer and light dose relationships for localized irradiation of a single blood vessel and are compared to the DLP required for vessel closure between 1- and 2- activation, between focal and broad-beam irradiation, and between verteporfin and a porphyrin dimer with high 2- cross section. Demonstration of reciprocity over a wide range of DLP is important for further development of focal PDT treatments, such as the targeting of feeder vessels in 2- PDT of AMD.

We have undertaken a study of the dynamics of CdSe/ZnS quantum dots in the blood vessels of the
chicken embryo chorioallantoic membrane (CAM). We show proof of principle that fluorescence correlation
spectroscopy can be used in this system to determine the concentrations and hydrodynamic radii of quantum dot
solutions micro-injected into the CAM.

Complete blood vessel occlusion is required for the treatment of age-related macular degeneration (AMD). AMD is the leading cause of blindness in developed countries and current treatment regimes have potential to cause collateral damage, or do not remove pre-existing unwanted vasculature. It has been proposed that two-photon excitation (TPE) photodynamic therapy (PDT) can be applied to cause local blood vessel occlusion without damaging surrounding retinal tissues. The in ovo chicken chorioallantoic membrane (CAM) is used as the model for vascularization in the wet form of AMD; novel techniques for the utilization of the CAM are reported. Complete occlusion of CAM vessels ~15 µm in diameter is achieved using the clinically approved photosensitizer Verteporfin (Visudyne®, QLT, Incorporated, Vancouver, British Columbia, Canada) and TPE activation. The average and peak irradiances used for treatment are 3.3×106 W/cm2 and 3.7×1011 W/cm2, respectively. A total fluence of 1.1×108 J/cm2 is the dosage required for successful occlusion, and it is expected that for optimal conditions it will be much less. These results are the first proof-of-principle evidence in the literature that indicate TPE-PDT can be used to occlude small blood vessels. Further investigation will help determine the utility of TPE-PDT for treating wet AMD, perhaps through targeting feeder vessels.

Photodynamic therapy (PDT) using verteporfin is widely used for treatment of age related macular degeneration (AMD).
Due to non-perfect selectivity of the drug accumulation in the neovasculature some collateral damage to healthy tissue
arises during the treatment. Damage to healthy structures in the eye is always a concern because of a high probability of
reducing visual acuity. Two-photon (2-&ggr;) photodynamic therapy potentially offers much higher treatment selectivity than
its one-photon (1-&ggr;) counterpart. By utilizing focused light for 2-&ggr; excitation, treatment volumes on the order of
microliters can be achieved thus maximizing localized insult to abnormal blood vessels and sparing healthy tissue. We
propose that 2-&ggr; photodynamic therapy will be valuable in the treatment of choroidal neovascularization secondary to
age related macular degeneration as well as other conditions. To ascertain feasibility of 2-&ggr; photodynamic therapy we
measured 2-&ggr; spectrum and cross sections of verteporfin (80 GM at 940 nm, 1 GM = 10<sup>-50</sup> cm<sup>4</sup>s/photon), chlorin e6 (14
GM at 800 nm) and tetrasulfonated aluminum phthalocyanine (140 GM at 900 nm) and investigated their <i>in vitro</i>
efficiency under 2-&ggr; excitation. Only verteporfin demonstrated cell kill under the used irradiation parameters (average
light intensity 9.1 mW, wavelength 850 nm, total light dose 6900 J/cm<sup>2</sup>). Dorsal skinfold window chamber model in
mouse was used to test efficiency of 2-&ggr; PDT with verteporfin <i>in vivo</i>. Although we were able to induce photodynamic
damage to a blood vessel using 1-&ggr; excitation, 2-&ggr; excitation resulted in no visible damage to irradiated blood vessel. The most probable reason is low efficiency of verteporfin as a 2-&ggr; photosensitizer. We also report 2-&ggr; spectrum of new
photosensitizer, HCC4 (4300 GM at 830 nm), specifically designed for efficient 2-&ggr; excitation.

The spectroscopy and photochemistry of protoporphyrin IX (PpIX) in ethanol and in Triton X-100 micelle solution and Verteporfin in methanol and Triton X-100 micelle solution have been examined using nea infrared two-photon excitation (TPE). TPE will allow photodynamic therapy with highly localized light dosage. For PpIX, we have determined that the photochemistry subsequent to TPE is very similar to that found for one-photon excitation. Moreover, the photoproducts observed possess very intense two-photon excitation fluorescence spectra, which allows their detection at low relative concentrations. Verteporfin displays photodynamic behavior in methanol similar to that of PpIX in ethanol. However, in micelle solution Verteporfin exhibits photodynamic behavior indicative of two sensitizer populations, excimers and monomers. Photochemical models are presented.

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